CN111849887A - Autologous fat three-dimensional gel and preparation method and application of SVF stem cells thereof - Google Patents
Autologous fat three-dimensional gel and preparation method and application of SVF stem cells thereof Download PDFInfo
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- APKFDSVGJQXUKY-INPOYWNPSA-N amphotericin B Chemical compound O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1/C=C/C=C/C=C/C=C/C=C/C=C/C=C/[C@H](C)[C@@H](O)[C@@H](C)[C@H](C)OC(=O)C[C@H](O)C[C@H](O)CC[C@@H](O)[C@H](O)C[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 APKFDSVGJQXUKY-INPOYWNPSA-N 0.000 claims description 7
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0667—Adipose-derived stem cells [ADSC]; Adipose stromal stem cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/35—Fat tissue; Adipocytes; Stromal cells; Connective tissues
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Abstract
The invention relates to a preparation method of autologous fat three-dimensional gel and SVF stem cells thereof, which comprises the following steps: 1) cleaning the collected adipose tissues by using a tissue cleaning solution until no blood residue exists, centrifuging the cleaned adipose tissues, removing supernatant, and shearing the cleaned adipose tissues to obtain fat particles; 2) adding fat tissue digestive juice into the fat particles obtained in the step 1) for primary digestion, adding trypsin for secondary digestion, centrifuging to remove supernatant after digestion is completed, and collecting precipitate; 3) cleaning the precipitate obtained in the step 3) by using a PBS solution, re-suspending the precipitate by using normal saline, screening to remove large tissues, and centrifuging to obtain autologous fat three-dimensional gel and SVF stem cells thereof; the adipose-derived mesenchymal stem cells obtained by the method have high yield, high purity and good activity, and the problems of low extraction rate, low purity and the like of the existing method for obtaining adipose-derived mesenchymal stem cells are solved.
Description
[ technical field ]
The invention belongs to the technical field of biology, and particularly relates to a preparation method and application of autologous fat three-dimensional gel and SVF stem cells thereof.
[ background art ]
The adipose-derived mesenchymal stem cell is a stem cell with the multipotentiality of multi-way differentiation and is separated from adipose tissues. In recent years, adipose-derived stem cells have become one of the most popular stem cells for tissue engineering and regenerative medicine. The autologous adipose-derived stem cells have wide sources, can secrete a large amount of growth factors such as epidermis, endothelium, blood vessels, fibrillization and the like, can promote the synthesis of human collagen, increase the blood vessels, are easy to survive, change the texture of human skin, reduce facial spots, fade facial wrinkles and improve facial depression, and thus, the change of facial rejuvenation is achieved.
Adipose tissues contain a cell with the potential of multidirectional differentiation, namely adipose tissue vascular stromal cells (SVF), wherein the SVF comprises adipose Mesenchymal Stem Cells (MSC), the biological properties of the SVF are similar to those of bone marrow mesenchymal stem cells, and the SVF can differentiate towards various cells such as fat, bone, cartilage, muscle endothelium, hematopoiesis, liver, pancreatic islet, nerve and the like. The fat tissue has rich storage in human body, easy acquisition and small wound. The amount of adipose tissue is much higher than the amount of bone marrow tissue for the same mass or volume of adipose tissue and bone marrow tissue. Therefore, the mesenchymal stem cells can replace a part of bone marrow mesenchymal stem cells, and have wider clinical application prospect. The adipose-derived mesenchymal stem cells are generally obtained by extracting human adipose tissues, and the applicable adipose-derived mesenchymal stem cells can be obtained only by performing separation treatment after the adipose tissues are extracted, but the existing treatment method has low extraction rate and low purity and activity of the obtained cells.
[ summary of the invention ]
The invention aims to solve the defects and provide a preparation method of autologous adipose three-dimensional gel and SVF stem cells thereof, wherein the obtained adipose-derived mesenchymal stem cells have high yield, high purity and good activity, and the problems of low extraction rate, low purity and the like of the conventional method for obtaining adipose-derived mesenchymal stem cells are solved.
In order to realize the purpose, the preparation method of the autologous fat three-dimensional gel and the SVF stem cells thereof comprises the following steps:
1) cleaning the collected adipose tissues by using a tissue cleaning solution until no blood residue exists, centrifuging the cleaned adipose tissues, removing supernatant, and shearing the cleaned adipose tissues to obtain fat particles;
2) adding fat tissue digestive juice into the fat particles obtained in the step 1) for primary digestion, adding trypsin for secondary digestion, centrifuging to remove supernatant after digestion is completed, and collecting precipitate;
3) and (3) cleaning the precipitate obtained in the step 3) by using a PBS solution, re-suspending the precipitate by using normal saline, screening to remove large tissues, and centrifuging to obtain the autologous fat three-dimensional gel and the SVF stem cells thereof.
Further, in the step 1), the tissue washing solution comprises physiological saline, amphotericin B, gentamicin sulfate and erythrocyte lysate.
Further, in step 1), the source of the autologous adipose tissue includes, but is not limited to, infrapatellar fat pad in the joint, medial thigh adipose tissue near the surgical joint site, or abdominal fat.
Further, in step 2), the fat tissue digestive juice comprises dnase, collagenase type I and collagenase type iii.
Further, the step 3) is followed by a culturing step, specifically, the autologous fat three-dimensional gel obtained in the step 3) and the SVF stem cells thereof are added into a cell culture solution and placed in an incubator for standing culture, the culturing condition is 37 ℃, the culturing time is 5% CO2, and the culturing time is 7-14 days.
Furthermore, the invention also provides an application of the autologous fat three-dimensional gel and SVF stem cells thereof as active ingredients in preparing medicines for improving mitochondrial damage of myocardial cells.
Compared with the prior art, the invention provides the autologous fat three-dimensional gel and the preparation method of the SVF stem cells thereof, which can fully digest adipose tissues and obtain rich nucleated cells by processing the adipose tissues through digestion, cleaning and the like, so that the obtained adipose mesenchymal stem cells have high yield, high purity and good activity, are favorable for clinical application, and solve the problems of low extraction rate, low purity and the like of the existing method for obtaining adipose mesenchymal stem cells.
[ detailed description of the invention ]
The invention is further illustrated below with reference to specific examples:
example 1:
a preparation method of autologous fat three-dimensional gel and SVF stem cells thereof comprises the following steps:
1) cleaning the collected adipose tissues by using a tissue cleaning solution until no blood residue exists, centrifuging the cleaned adipose tissues, removing supernatant, and shearing the cleaned adipose tissues to obtain fat particles; the tissue cleaning fluid comprises normal saline, amphotericin B, gentamicin sulfate and erythrocyte lysate;
2) adding fat tissue digestive juice into the fat particles obtained in the step 1) for primary digestion, adding trypsin for secondary digestion, centrifuging to remove supernatant after digestion is completed, and collecting precipitate; the fat tissue digestive fluid comprises the following components in concentration: 0.06-0.07 mg/mL of DNase, 0.3-0.45 mg/mL of collagenase type I and 0.5-0.65 mg/mL of collagenase type III, wherein a solvent adopts a DMEM medium containing double antibodies; the double antibodies in the DMEM medium containing the double antibodies are 0.6-1.2 mug/mL gentamicin sulfate and 0.6-1.2 mug/mL amphotericin B.
3) And (3) cleaning the precipitate obtained in the step 3) by using a PBS solution, re-suspending the precipitate by using normal saline, screening to remove large tissues, and centrifuging to obtain the autologous fat three-dimensional gel and the SVF stem cells thereof.
The autologous fat three-dimensional gel and the SVF stem cells thereof can be used as active ingredients to prepare medicines for improving the mitochondrial injury of myocardial cells.
Example 2:
a preparation method of autologous fat three-dimensional gel and SVF stem cells thereof comprises the following steps:
1) cleaning the collected adipose tissues by using a tissue cleaning solution until no blood residue exists, centrifuging the cleaned adipose tissues, removing supernatant, and shearing the cleaned adipose tissues to obtain fat particles; the preparation method of the tissue cleaning fluid comprises the following steps: uniformly mixing normal saline, gentamicin sulfate, amphotericin B and erythrocyte lysate, wherein the concentration of gentamicin sulfate is 20-24 mug/mL, the concentration of amphotericin B is 12.5-14 mug/mL, and the erythrocyte lysate accounts for 53-55% of the total volume.
2) Adding fat tissue digestive juice into the fat particles obtained in the step 1) for primary digestion, adding trypsin for secondary digestion, centrifuging to remove supernatant after digestion is completed, and collecting precipitate; the fat tissue digestive juice comprises DNA enzyme, collagenase type I and collagenase type III;
3) and (3) cleaning the precipitate obtained in the step 3) by using a PBS solution, re-suspending the precipitate by using normal saline, screening to remove large tissues, and centrifuging to obtain the autologous fat three-dimensional gel and the SVF stem cells thereof.
Example 3:
a preparation method of autologous fat three-dimensional gel and SVF stem cells thereof comprises the following steps:
1) cleaning the collected adipose tissues by using a tissue cleaning solution until no blood residue exists, centrifuging the cleaned adipose tissues, removing supernatant, and shearing the cleaned adipose tissues to obtain fat particles; the tissue cleaning fluid comprises normal saline, amphotericin B, gentamicin sulfate and erythrocyte lysate; sources of autologous adipose tissue include, but are not limited to, intra-articular infrapatellar fat pads, medial thigh adipose tissue near the surgical joint site, or abdominal fat;
2) adding fat tissue digestive juice into the fat particles obtained in the step 1) for primary digestion, adding trypsin for secondary digestion, centrifuging to remove supernatant after digestion is completed, and collecting precipitate; the fat tissue digestive juice comprises DNA enzyme, collagenase type I and collagenase type III;
3) cleaning the precipitate obtained in the step 3) by using a PBS solution, re-suspending the precipitate by using normal saline, screening to remove large tissues, and centrifuging to obtain autologous fat three-dimensional gel and SVF stem cells thereof;
4) adding the autologous fat three-dimensional gel obtained in the step 3) and the SVF stem cells thereof into a cell culture solution, placing the cell culture solution into an incubator for standing culture, wherein the culture condition is 37 ℃ and 5% CO2 incubator, and the culture time is 7-14 days.
The autologous fat three-dimensional gel and the SVF stem cells thereof can be used as active ingredients to prepare medicines for improving the mitochondrial injury of myocardial cells.
The present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents and are included in the scope of the present invention.
Claims (6)
1. The preparation method of the autologous fat three-dimensional gel and the SVF stem cells thereof is characterized by comprising the following steps:
1) cleaning the collected adipose tissues by using a tissue cleaning solution until no blood residue exists, centrifuging the cleaned adipose tissues, removing supernatant, and shearing the cleaned adipose tissues to obtain fat particles;
2) adding fat tissue digestive juice into the fat particles obtained in the step 1) for primary digestion, adding trypsin for secondary digestion, centrifuging to remove supernatant after digestion is completed, and collecting precipitate;
3) and (3) cleaning the precipitate obtained in the step 3) by using a PBS solution, re-suspending the precipitate by using normal saline, screening to remove large tissues, and centrifuging to obtain the autologous fat three-dimensional gel and the SVF stem cells thereof.
2. The method for preparing autologous fat three-dimensional gel and SVF stem cells thereof according to claim 1, wherein: in the step 1), the tissue cleaning solution comprises normal saline, amphotericin B, gentamicin sulfate and erythrocyte lysate.
3. The method for preparing autologous fat three-dimensional gel and SVF stem cells thereof according to claim 1, wherein: in step 1), the source of the autologous adipose tissue includes, but is not limited to, infrapatellar fat pad in joints, medial thigh adipose tissue near surgical joint sites or abdominal fat.
4. The method for preparing autologous fat three-dimensional gel and SVF stem cells thereof according to claim 1, wherein: in step 2), the fat tissue digestive juice comprises DNase, collagenase type I and collagenase type III.
5. The method for preparing autologous fat three-dimensional gel and SVF stem cells thereof according to claim 1, wherein: and 3) a culture step is further included after the step 3), specifically, the autologous fat three-dimensional gel obtained in the step 3) and the SVF stem cells thereof are added into a cell culture solution and placed in an incubator for standing culture, the culture conditions are 37 ℃ and 5% CO2 incubator, and the culture time is 7-14 days.
6. The autologous fat three-dimensional gel and SVF stem cells thereof according to any of claims 1 to 5, as active ingredients for use in improving mitochondrial damage of cardiomyocytes.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113416694A (en) * | 2021-07-21 | 2021-09-21 | 江苏瑞思坦生物科技有限公司 | Method for efficiently obtaining adipose-derived mesenchymal stem cells from trace fat |
| CN114832016A (en) * | 2022-04-19 | 2022-08-02 | 芙普瑞生物细胞科学(苏州)有限公司 | Adipose SVF cell preparation and application thereof |
-
2020
- 2020-08-05 CN CN202010777925.2A patent/CN111849887A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113416694A (en) * | 2021-07-21 | 2021-09-21 | 江苏瑞思坦生物科技有限公司 | Method for efficiently obtaining adipose-derived mesenchymal stem cells from trace fat |
| CN114832016A (en) * | 2022-04-19 | 2022-08-02 | 芙普瑞生物细胞科学(苏州)有限公司 | Adipose SVF cell preparation and application thereof |
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